This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Endocytic transport is a key mechanism for controlling the subcellular distribution of free cholesterol and the endocytic recycling compartment (ERC) is an important organelle that stores cholesterol and regulates its transport. Rab11, which regulates transport via the ERC, regulates the exit of cholesterol from this organelle. EHD1, a member of the C-terminal EH-domain family of proteins that regulates recycling and coordinates transport via the Rab11 pathway, has also been reported to play a role in free cholesterol transport through the ERC. To directly assess the role of EHD1 on cholesterol homeostasis and cellular distribution, we utilized mouse embryonic fibroblasts derived from EHD1 knockout mice (MEF -/-). Surprisingly, these cells displayed reduced levels of cellular free and esterified cholesterol, an effect that could be rescued by overexpression of wild-type EHD1. To understand the reduction in intracellular cholesterol in the absence of EHD1, we turned our focus to low density lipoprotein (LDL) and its receptor, LDLR, a major source of cellular cholesterol intake. We observed higher levels of LDLR on the plasma membrane of MEF -/- cells, yet LDL itself was internalized at a slower rate in these cells. Furthermore, in cells lacking EHD1, lipid droplets appeared greatly reduced in size, suggesting that less esterified cholesterol and triglycerides were packaged in lipid droplets. Two-hybrid binding assays and NMR spectroscopy suggest that the EH-domain of EHD1 interacts with Epsin, a well-characterized component of the endocytic pathway that contains NPF motifs and regulates receptor internalization. Our data indicates EHD1 affects cholesterol homeostasis and lipid droplet biogenesis by controlling internalization of LDL receptor, possibly through interactions with NPF-containing endocytic regulators such as epsin.